Vitamin A (retinol) and its derivatives belong to a class of compounds known as retinoids. Retinoids are an important class of signalling molecules that are involved in controlling many important biological pathways from embryogenesis through to adult homeostasis and many aspects of stem cell development, such as, stem cell proliferation, differentiation and apoptosis.
Retinoids are structurally and/or functionally related to vitamin A; and many possess biological activity including all-trans-retinoic acid (ATRA). ATRA is the most abundant endogenous retinoid and has been widely studied for many years; ATRA isomerises under physiological and experimental conditions, with different isomers activating different receptors, thus accounting for the variety of biological effects observed with these small molecules.
Due to the ability of retinoids to control differentiation and apoptosis in both normal and tumour cells, they have the potential to act as chemopreventative and chemotherapeutic agents, although toxicity has prevented widespread use.
However, ATRA exhibits poor stability, in particular upon exposure to light. ATRA compounds isomerise and degrade upon exposure to light. To overcome this, efforts are made to store and work with ATRA in the dark, but such precautions increase the cost associated with working with ATRA, and do not entirely mitigate the problem. Furthermore, as ATRA is liable to photoisomerisation and degradation upon storage, it is difficult to predict accurately the amount of active compound administered in a single dose. Efforts have been made to overcome the problems associated with ATRA by synthesising stable retinoid compounds. It is generally believed that ATRA is susceptible to photoisomerisation due to its conjugated linker group.
International Patent application No. PCT/GB2007/003237 (WO 2008/025965) disclosed new retinoid compounds which exhibited good stability and induced cell differentiation.
One compound of particular interest was EC23®, which is/was marketed by Reinnervate:
EC23® generally exhibits good stability exposed to light, as well as exhibiting good stability upon storage. EC23® is also found to not be susceptible to metabolic degradation, and thus may have a relatively long associated half-life in the human or animal body. However, EC23® is only weakly fluorescent, and requires UV excitation, which may be damaging to biological samples.
Fluorescence imaging has rapidly become a powerful tool for investigating biological processes, particularly in living cells where cellular events may be observed in their physiological contexts. The development of single-molecule visualisation techniques has greatly enhanced the usefulness of fluorescence microscopy for such applications, enabling the tracking of proteins and small molecules in their endogenous environments.
Fluorescence is a form of luminescence in which a substance that has absorbed light or other electromagnetic radiation emits light from electronically excited states. In fluorescence, the emitted light is usually of a longer wavelength (and lower energy) than the absorbed light. This phenomenon is known as Stokes shift, and is attributed to the loss of energy, usually via vibrational relaxation to the lowest energy level of the first excited state (S1), before an absorbed photon is emitted. The quantum yield gives the efficiency of the fluorescence process: it is defined as the ratio of the number of photons emitted to the number of photons absorbed (maximum value=1, i.e. every absorbed photon results in an emitted photon). Fluorescence decay is generally exponential and the fluorescence lifetime refers to the measure of the half-life of a molecule remaining in an excited state before undergoing relaxation back to the ground state. In phosphorescence, a longer excited state lifetime is observed, followed by radiative decay (i.e. photon emission) from an excited triplet state.
Doxorubicin is a chemotherapeutic drug used in the treatment of a wide range of cancers, including leukaemia, Hodgkin's lymphoma, bladder, breast, stomach, lung, ovarian, and thyroid cancers. The amphiphilic and amphoteric nature of the molecule means that the drug is able to bind to both cell membranes and proteins.

Due to the inherent fluorescence of the compound, doxorubicin has also become a popular research tool in the field of fluorescence imaging, and its distribution has accordingly been visualised in various cells and tissues. Since the fluorescence intensity of doxorubicin was found to be dependent on its concentration and microenvironment, the intracellular uptake and trafficking of the drug in ovarian carcinoma A2780 cells was able to be characterised by taking into account its interaction with cellular components such as DNA, histones, and phospholipids.
At present, doxorubicin is the only known small molecule possessing intrinsic fluorescence emission along with significant biological activity. Thus, if fluorescence could be incorporated into a small molecule modulator of stem cell development, this would in itself constitute a powerful probe, and would negate the need for the use of fluorescent dyes, proteins, and quantum dots. In particular, the use of live-cell tracking techniques would provide invaluable information concerning cellular uptake and localisation, thereby offering new insights into retinoid activity and metabolism. Furthermore, since it would no longer be necessary to attach a large fluorescent entity to the molecule of interest, the latter may be followed in the physiological context of its natural environment. In addition, it may also be advantageous to generate an inert fluorescent probe that may have useful fluorescent properties.
Therefore, for improved fluorescence imaging, there is a need for a novel fluorophore that exhibits good storage stability, and is not susceptible to metabolic degradation, thus having a relatively long associated half-life in the body. Thus, an object of the present invention is to provide a stable fluorescent retinoid.